High-pressure liquid chromatography (HPLC) solvent delivery systems are used to source single-component liquids or mixtures of liquids (both known as “mobile phase”) at pressures which can range from substantially atmospheric pressure to pressures on the order of ten thousand pounds per square inch. These pressures are required to force the mobile phase through the fluid passageways of a stationary phase support, where separation of dissolved analytes can occur. The stationary phase support may comprise a packed bed of particles, a membrane or collection of membranes, a porous monolithic bed, or an open tube. Often, analytical conditions require the mobile phase composition to change over the course of the analysis (this mode being termed “gradient elution”). In gradient elution, the viscosity of the mobile phase may change and the pressure necessary to maintain the required volumetric flow rate will change accordingly.
In liquid chromatography, the choice of an appropriate separation strategy (including hardware, software, and chemistry) results in the separation of an injected sample mixture into its components, which elute from the column in reasonably distinct zones or “bands”. As these bands pass through a detector, their presence can be monitored and a detector output (usually in the form of an electrical signal) can be produced. The pattern of analyte concentration within the eluting bands, which can be represented by means of a time-varying electrical signal, gives rise to the nomenclature of a “chromatographic peak”. Peaks may be characterized with respect to their “retention time”, which is the time at which the center of the band transits the detector, relative to the time of injection (i.e. time-of-injection is equal to zero). In many applications, the retention time of a peak is used to infer the identity of the eluting analyte, based upon related analyses with standards and calibrants. The retention time for a peak is strongly influenced by the mobile phase composition, and by the accumulated volume of mobile phase which has passed over the stationary phase.
The utility of chromatography relies heavily on run-to-run reproducibility, such that standards or calibrants can be analyzed in one set of runs, followed by test samples or unknowns, followed by more standards, in order that confidence can be had in the resulting data. Known pumping systems exhibit some non-ideal characteristics which result in diminished separation performance and diminished run-to-run reproducibility. Among the non-ideal pump characteristics exhibited in known pumping systems are, generally, fluctuations in solvent composition and/or fluctuations in volumetric flow rate.
Volumetric flow fluctuations present in known HPLC pumping systems disadvantageously cause retention time(s) to vary for a given analyte. That is, the amount of time that an analyte is retained in the stationary phase fluctuates undesirably as a function of the undesirable volumetric flow fluctuations. This creates difficulties in inferring the identity of a sample from the retention behavior of the components. Volumetric flow fluctuations from individual pumps can result in fluctuations in solvent composition when the output of multiple pumps is summed to provide a solvent composition.
Fluctuations in solvent composition present in known HPLC pumping systems can disadvantageously result in interactions with the system's analyte detector and produce perturbations which are detected as if they arose from the presence of a sample. In effect, an interference signal is generated. This interference signal is summed with the actual signal attributable to the analyte, producing errors when the quantity of an unknown sample is calculated from the area of the eluting sample peak.
The prior art is replete with techniques and instrument implementations aimed at controlling solvent delivery and minimizing perturbations in the output of delivery systems for analytical instrumentation. Myriad pump configurations are known which deliver fluid at high pressure for use in applications such as liquid chromatography. Known pumps, such as one disclosed in U.S. Pat. No. 4,883,409 (“the '409 patent”) incorporate at least one plunger or piston which is reciprocated within a pump chamber into which fluid is introduced. A controlled reciprocation frequency and stroke length of the plunger within the pump chamber determines the flow rate of fluid output from the pump. However, the assembly for driving the plunger is an elaborate combination of elements that can introduce undesirable motion in the plunger as it is driven, which motion makes it difficult to precisely control the solvent delivery system output and results in what is termed “noise” or detectable perturbations in a chromatographic baseline. Much of this noise does not result from random statistical variation in the system, rather much of it is a function of a mechanical “signature” of the pump. Mechanical signature is correlated to mechanically related phenomena such as anomalies in ball and screw drives, gears, and/or other components used in the pump to effect the linear motion that drives the piston(s), or it is related to higher level processes or physical phenomena such as the onset or completion of solvent compression, or the onset of solvent delivery from the pump chamber.
Typical systems known for delivery of liquids in liquid chromatography applications, such as disclosed in the '409 patent and further in U.S. Pat. No. 5,393,434, implement dual piston pumps having two interconnected pump heads each with a reciprocating plunger. The plungers are driven with a predetermined phase difference in order to minimize output flow variations. Piston stroke length and stroke frequency can be independently adjusted when the pistons are independently, synchronously driven. Precompression can be effected in each pump cylinder in any given pump cycle to compensate for varying fluid compressibilities in an effort to maintain a substantially constant system pressure and output flow rate.
There are two widely used means to create gradient HPLC pumps. The solvents can be blended on the intake side of the pump. This is known in the art as low pressure gradient mixing. The alternative is the use of so-called high pressure gradient systems in which each individual solvent is delivered by a separate pump.
The fundamental scalar of all forms of gradient chromatography is the void volume of the separation column. The void volume of an HPLC column is the sum of the inter and intra particle volumes of the column that are filled with mobile phase. The void volume is the minimum volume required to elute an unretained solute. The gradient delay volume is the volume of the mobile phase delivered from the time the gradient is initiated to when the change in composition first arrives at the column. The delay volume is the volumetric overhead of the gradient solvent delivery system; it adds to the time required to complete the separation and to prepare the column for the next injection. The delay volume should be minimized and ideally should be no more than two times larger than the void volume of the column.
When two or more high pressure pumps are combined to form a gradient solvent delivery system, their outputs are combined with the resulting possibility that there can be fluidic cross talk between the high pressure pumps during their individual piston crossovers. One prior approach to avoid fluidic cross talk has been the use of pulse dampeners within the gradient solvent delivery system as shown in FIG. 1.
When individual pulse dampeners are placed up-stream from where output of the solvents meet and the total flow is small relative to the volume of the pulse dampeners, there will be significant crosstalk between the pumps since the two pumps are not synchronous in their respective piston crossovers. This crosstalk occurs because the fluid contained in the off line pulse dampener can be compressed making it the low impedance path for the on-line pump. As such, this up-stream placement of the pulse dampeners results in a compromised flow rate and composition. The result of this fluidic crosstalk is shown in FIG. 2, which plots the delivery of a gradient marker from a solvent delivery system configured as shown in FIG. 1 at a low flow rate. As shown in FIG. 2, no gradient deliveries are identical and none correspond to the programmed gradient. This results in unsatisfactory and unpredictable separations which cannot be reproduced.
In an alternative prior art approach, a capillary restrictor is used to generate backpressure to energize the pulse dampeners. A capillary of fixed length and internal diameter provides sufficient backpressure to restrict, but unfortunately not prevent backflow, over narrow ranges of flow rates.
A further approach to the use of pulse dampeners is to position a pulse dampener downstream from the common mixing tee. While this approach is useful in gradient systems having large volumes, smaller scale volumes are problematic. The positioning of a pulse dampener after the common mixing tee greatly increases the delay volume within the gradient systems. Pulse dampeners are scaled to a specific and limited flow rate range as they typically combine resistance to flow and a captive capacitive volume of the mobile phase. The requirements of effective pulse dampening and minimizing delay volume will conflict as the scale of the HPLC system with respect to column volume and volumetric flow rate is reduced.